Methods for producing biological materials

ABSTRACT

Methods of producing biological materials from cells and organisms are provided. Aspects of the methods include modulating the stress conditions of the cells and/or organism to produce biological materials having one or more desired properties. In certain aspects, the cell or organism is evaluated to detect the presence or absence of a stressed phenotype, wherein an unstressed phenotype may be produced before the cell or organism produces the biological material of interest. The biological materials produced from such cells and organisms may be used for a variety of applications, including therapeutic, research, and other applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 61/674,232filed Jul. 20, 2012; the disclosure of which is herein incorporated byreference.

INTRODUCTION

A variety of useful biological materials may be produced by organisms,including therapeutics (e.g. peptides and antibodies), research tools,and nutritional products. Further, certain cells that are harvested orderived from organisms may themselves be used to produce a range ofuseful biological materials. However, adequately maintaining andculturing the living organisms and cells to produce the desiredbiological materials is often a difficult problem. Current approachesmay lead to the production of biological materials that have one or moreproblems, such as suboptimal safety, efficacy, and/or yield.

SUMMARY

Methods of producing biological materials from cells and organisms areprovided. Aspects of the methods include modulating the stressconditions of the cells and/or organism to produce biological materialshaving one or more desired properties. In certain aspects, the cell ororganism is evaluated to detect the presence or absence of a stressedphenotype, wherein an unstressed phenotype may be produced before thecell or organism produces the biological material of interest. Thebiological materials produced from such cells and organisms may be usedfor a variety of applications, including therapeutic, research, andother applications.

Methods of the present disclosure include producing a biological productfrom a cell, the methods involving culturing the cell containing nucleicacid encoding at least a portion of the biological product, underconditions allowing for expression of the biological product; evaluatingthe cell to identify the presence or absence of a stressed phenotype;and recovering the biological product.

Methods of the present disclosure also include producing a biologicalproduct from an organism, the methods involving evaluating an organismto identify the presence or absence of an unstressed phenotype;extracting a plurality of cells from the organism; introducing nucleicacid encoding at least a portion of the biological product into theplurality of cells; culturing the cells under conditions allowing forexpression of the biological product; and recovering the biologicalproduct.

The methods of the present disclosure further include methods forproducing a product from an organism having an unstressed phenotype, themethods involving evaluating an organism to identify the presence of astressed phenotype; producing an unstressed phenotype in the organism;maintaining the organism under conditions allowing for production of theproduct; and recovering the product.

The methods for determining the presence or absence of a stressedphenotype may vary, and may include visual inspection, behavioralassessment, genomic assessment, proteomic assessment, and the like. Incells or organisms identified as having a stressed phenotype, anunstressed phenotype may be produced by a variety of means, includingbut not limited to changing the nutritional intake (e.g., type of food,quantity of food, etc.), changing the subject's environment (e.g.,temperature, exposure to light, etc.), and/or administering an agent toreduce stress. Stress reducing agents of interests include, but are notlimited to, small organic compounds and peptides.

The biological products produced by the methods of the presentdisclosure may also vary. Biological products of interest include, butare not limited to, recombinant therapeutic proteins, viruses (e.g.recombinant viruses for gene therapy), vaccines, antibodies, proteinsand peptides (e.g., enzymes, growth factors, etc.), polysaccharides,nucleic acids (including DNA and RNA), cells, and nutritional products.Methods of the present disclosure may be used in conjunction withseveral different production techniques known in the art, such as theproduction of biological products using cells in a bioreactor (e.g.,mammalian, yeast, bacteria, and/or insect cells), methods involving theuse of transgenic animals (e.g. goats or chickens), methods involvingthe use of transgenic plants (e.g., tobacco, seeds or moss), and othermethods known to those of skill in the art.

In practicing the subject methods, cells of interest include, but arenot limited to, CHO cells, COS cells, NS0 cells, SP2/0 cells, and YB2/0cells. Organisms of interest include, but are not limited to:prokaryotes, e.g. bacteria, archaea and cyanobacteria; and eukaryotes,e.g. members of the kingdom protista, such as flagellates, amoebas andtheir relatives, amoeboid parasites, ciliates and the like; members ofthe kingdom fungi, such as slime molds, acellular slime molds, cellularslime molds, water molds, true molds, conjugating fungi, sac fungi, clubfungi, imperfect fungi and the like; plants, such as algae, mosses, andflowering plants, both monocots and dicots; and animals, includingvertebrates, including fishes, birds, and mammals, e.g. rodents,primates, including humans, and the like.

DETAILED DESCRIPTION

Methods of producing biological materials from cells and organisms areprovided. Aspects of the methods include modulating the stressconditions of the cells and/or organism to produce biological materialshaving one or more desired properties. In certain aspects, the cell ororganism is evaluated to detect the presence or absence of a stressedphenotype, wherein an unstressed phenotype may be produced before thecell or organism produces the biological material of interest. Thebiological materials produced from such cells and organisms may be usedfor a variety of applications, including therapeutic, research, andother applications.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andexemplary methods and materials may now be described. Any and allpublications mentioned herein are incorporated herein by reference todisclose and describe the methods and/or materials in connection withwhich the publications are cited. It is understood that the presentdisclosure supersedes any disclosure of an incorporated publication tothe extent there is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “abiological product” includes a plurality of such biological products andreference to “the cell” includes reference to one or more cells, and soforth.

It is further noted that the claims may be drafted to exclude anyelement which may be optional. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely”, “only” and the like in connection with the recitation of claimelements, or the use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.To the extent such publications may set out definitions of a term thatconflict with the explicit or implicit definition of the presentdisclosure, the definition of the present disclosure controls.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

As summarized above, aspects of the invention include methods ofproducing biological materials from cells and organisms, with suchmethods involving modulating the stress conditions of the cells and/ororganism to produce biological materials having one or more desiredproperties.

The terms “biological material” and “biological product” may be usedinterchangeably herein, and refer to any product or material that isproduced by a cell or organism, including the cell or organism itself.The biological material may or may not be a natural product of the cellor organism, so long as the product or material is produced by the cellor organism. Exemplary biological materials include, but are not limitedto, recombinant therapeutic proteins, viruses (e.g. recombinant virusesfor gene therapy), vaccines, antibodies (e.g. IgG antibodies, monoclonalantibodies, and the like), proteins and peptides (e.g., enzymes, growthfactors, etc.), polysaccharides, nucleic acid including DNA and RNA,cells, and nutritional products for consumption by an organism (e.g.eggs, livestock). As used herein, a biological product that is producedby a cell or organism and subsequently modified or altered in one ormore ways (e.g. glycosylation, radiolabeling, and the like) is stillconsidered a biological product.

Thus, biological products may be viewed as those that are produced by acell and/or organism. Organisms referred to herein may varyconsiderably, where the term “organism” refers to a living oronce-living entity that may range in size and complexity from singlecell entities to complex multicellular species. As such, organisms ofinterest include, but are not limited to: single celled organisms, e.g.prokaryotes, and multicellular organisms, e.g., invertebrates andvertebrates, etc. Accordingly, organisms of interest include, but arenot limited to: prokaryotes, e.g. bacteria, archaea and cyanobacteria;and eukaryotes, e.g. members of the kingdom protista, such asflagellates, amoebas and their relatives, amoeboid parasites, ciliatesand the like; members of the kingdom fungi, such as yeasts, slime molds,acellular slime molds, cellular slime molds, water molds, true molds,conjugating fungi, sac fungi, club fungi, imperfect fungi and the like;plants, such as algae, mosses, liverworts, hornworts, club mosses,horsetails, ferns, herbs, shrubs, e.g. tobacco plants, gymnosperms andflowering plants, both monocots and dicots; and animals, includingsponges, members of the phylum cnidaria, e.g. jelly fish, corals and thelike, combjellies, worms, rotifers, roundworms, annelids, molluscs,arthropods, echinoderms, acorn worms, and vertebrates, includingreptiles, fishes, birds (e.g. chickens), snakes, and mammals, e.g.rodents, bovids (e.g. goats), primates, including humans, and the like.Organisms may be naturally occurring or non-naturally occurring (e.g.transgenic).

Accordingly, the “cells” referred to herein may also vary considerably,where the term “cell” refers to any cell harvested and/or derived fromone or more organisms. Where an organism is a single celled organism,the terms “cell” and “organism” may thus be used interchangeably.Moreover, the term “cell” may refer to both naturally occurring andnon-naturally occurring cells. Suitable cells include eukaryotic cells,such as a mammalian cell, an insect cell, a yeast cell; and prokaryoticcells, such as a bacterial cell.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like.

Suitable yeast cells include, but are not limited to, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonasreinhardtii, and the like.

Suitable prokaryotic cells include, but are not limited to, any of avariety of laboratory strains of Escherichia coli, Lactobacillus sp.,Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al.(1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemoreet al. (1995) Science 270:299-302. Examples of Salmonella strains whichcan be employed include, but are not limited to, Salmonella typhi and S.typhimurium. Suitable Shigella strains include, but are not limited to,Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically,the laboratory strain is one that is non-pathogenic. Non-limitingexamples of other suitable bacteria include, but are not limited to,Bacillus subtilis, Pseudomonas pudita, Pseudomonas aeruginosa,Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus,Rhodospirillum rubrum, Rhodococcus sp., and the like. In someembodiments, the cell is Escherichia coli.

In the context of the current invention, a cell and/or organism is usedto produce a biological material, where the material may vary greatlyfrom therapeutic products to nutritional products. The inventors havediscovered that cells and organisms used to produce biological materialsmay be stressed, which may lead to the production of biologicalmaterials that have one or more suboptimal properties, such assuboptimal safety, efficacy, and/or yield. Aspects of the methodsinclude modulating the stress conditions of the cells and/or organism toproduce biological materials having one or more desired properties,and/or the absence of one or more undesired properties (e.g. poor safetyprofile, poor efficacy, and the like).

In certain aspects, a biological product is produced by an unstressedcell or organism. The organism or cell may be acquired by any convenientmeans, including by purchasing from a commercial source that sells cellsor organisms. Once the cell or organism has been provided, the presenceor absence of a stress phenotype may be evaluated. Stress may manifestitself in a number of different phenotypic ways, ranging from outwardappearance (e.g., in some way deviating from a normal, wild-typeappearance) or in other ways, e.g., by changes in genomic and/orproteomic profiles. The cell and/or organism may be evaluated by one ormore ways, including but not limited to: appearance to behavior tocertain stressed genomic and/or proteomic profiles. A variety ofdifferent methods for evaluating stress phenotypes in cells and/ororganisms are known to those of skill in the art, including thosedescribed in National Research Council. (2008) Recognition andAlleviation of Distress in Laboratory Animals. Washington, D.C.: TheNational Academies Press; Moberg G P, Mench J A (2000) The Biology ofAnimal Stress. Basic Principles and Implications for Animal Welfare.Wallingford: CABI Publishing; Morton, D. B. and P. H. M. Griffiths.1985. Guidelines on the recognition of pain, distress and discomfort inexperimental animals and an hypothesis for assessment. Vet Record116(16):431-436; Kuntz, D. 2005. Molecular and evolutionary basis of thecellular stress response. Annual Review of Physiology. 67:225-257;Fulda, S., A. M. Gorman, O. Hori, and A. Samali. 2010. Cellular StressResponses: Cell Survival and Cell Death. International Journal of CellBiology. vol. 2010, Article ID 214074, 23 pages; the disclosures ofwhich are herein incorporated by reference.

Evaluation may include visual assessment of outward phenotypiccharacteristics, such as visual inspection of the appearance of the cellor organism, e.g., to determine the presence of stress by identifyingthe presence of one or more stress correlated appearancecharacteristics, as described in, for example, Garner, J. P., S. M.Weisker, B. Dufour, and J. A. Mench. 2004. Barbering (fur and whiskertrimming) by laboratory mice as a model of human trichotillomania andobsessive-compulsive spectrum disorder. Comparative Med 54(2):216-224;the disclosure of which is herein incorporated by reference.

Evaluation may, in certain embodiments, include behavioral assessment ofthe cell or organism, e.g., to determine the presence of stress byidentifying one or more stress correlated behavioral characteristics,such as is described in Abeyesinghe, S. M., C. M. Wathes, C. J. Nicol,and J. M. Randall. 2001. The aversion of broiler chickens to concurrentvibrational and thermal stressors. Appl Anim Behav Sci 73(3):199-215;Bayne, K. A. L., S. Dexter, and S. Suomi. 1992. A preliminary survey ofthe incidence of abnormal behavior in rhesus monkeys (Macaca mulatto)relative to housing condition. Lab Anim 22(5):38-44; Beck, K. D. and V.N. Luine. 2002. Sex differences in behavioral and neurochemical profilesafter chronic stress: Role of housing conditions. Physiol Behav75(5):661-673; Blanchard, R. J., C. R. McKittrick, and D. C. Blanchard.2001. Animal models of social stress: Effects on behavior and brainneurochemical systems. Physiol Behav 73(3):261-271; Crawley J N. 2000.What's Wrong with My Mouse? Behavioral Phenotyping of Transgenic andKnockout Mice. New York: Wiley-Liss; Crawley J N. 2003. Behavioralphenotyping of rodents. Comp Med 53:140-146; D'Aquila, P. S., P. Brain,and P. Willner. 1994. Effects of chronic mild stress on performance inbehavioural tests relevant to anxiety and depression. Physiol Behav56(5):861-867; Garner, J. P., C. L. Meehan, and J. A. Mench. 2003.Stereotypies in caged parrots, schizophrenia and autism: Evidence for acommon mechanism. Behav Brain Res 145(1-2):125-134; Harkin, A., T. J.Connor, J. M. O'Donnell, and J. P. Kelly. 2002. Physiological andbehavioral responses to stress: What does a rat find stressful? Lab Anim31(4):42-50; Mangiavacchi, S., F. Masi, S. Scheggi, B. Leggio, M. G. DeMontis, and C. Gambarana. 2001. Long-term behavioral and neurochemicaleffects of chronic stress exposure in rats. J Neurochem 79(6):1113-1121;Mayer, J. 2007. Use of behavior analysis to recognize pain in smallanimals. Lab Anim 36(6):43-48; Mineur, Y. S., D. J. Prasol, C. Belzung,and W. E. Crusio. 2003. Agonistic behavior and unpredictable chronicmild stress in mice. Behav Genet 33(5):513-519; Trullas, R. and P.Skolnick. 1993. Differences in fear motivated behaviors among inbredmouse strains. Psychopharmacology 111(3):323-331; Vogt, J. L., C. L.Coe, E. Lowe, and S. Levine. 1980. Behavioral and pituitary-adrenalresponse of adult squirrel monkeys to mother-infant separation.Psychoneuroendocrinology 5(3):181-190; the disclosures of which areherein incorporated by reference.

Evaluation may, in certain embodiments, include genomic assessment ofthe cell or organism, e.g., to determine the presence of stress byidentifying one or more stress correlated gene expressioncharacteristics (as may be readily identified by gene expressionanalysis), such as is described in Cook, M. N., R. W. Williams, and L.Flaherty. 2001. Anxiety-related behaviors in the elevated zero-maze areaffected by genetic factors and retinal degeneration. Behav Neurosci115(2):468-476; Mangiarini, L., K. Sathasivam, M. Seller, B. Cozens, A.Harper, C. Hetherington, M. Lawton, Y. Trottier, H. Lehrach, S. W.Davies, and G. P. Bates. 1996. Exon 1 of the HD gene with an expandedCAG repeat is sufficient to cause a progressive neurological phenotypein transgenic mice. Cell 87(3):493-506; Stenzel-Poore, M., S. C.Heinrichs, S. Rivest, G. F. Koob, and W. W. Vale. 1994. Overproductionof corticotrophin-releasing factor in transgenic mice: A genetic modelof anxiogenic behavior. J Neurosci 14(5 Pt 1):2579-2584; the disclosuresof which are herein incorporated by reference.

Evaluation may, in certain embodiments, include proteomic assessment ofthe cell or organism, e.g., to determine the presence of stress byidentifying one or more stress correlated protein expressioncharacteristics (as may be readily identified using protein expressionanalysis), as described in Hyun, D. H., S. S. Emerson, D. G. Jo, M. P.Mattson, and R. de Cabo. 2006. Calorie restriction up-regulates theplasma membrane redox system in brain cells and suppresses oxidativestress during aging. Proc Natl Acad Sci USA 103(52):19908-19912; Maier,S. F. and L. R. Watkins. 2005. Stressor controllability and learnedhelplessness: The roles of the dorsal raphe nucleus, serotonin, andcorticotropin-releasing factor. Neurosci Biobehav 29(4-5):829-841;Muglia, L., L. Jacobson, P. Dikkes, and J. A. Majzoub. 1995.Corticotropin-releasing hormone deficiency reveals major fetal but notadult glucocorticoid need. Nature 373(6513):427-432; Orr, T. E., J. L.Meyerhoff, E. H. Mougey, and B. N. Bunnell. 1990. Hyperresponsiveness ofthe rat neuroendocrine system due to repeated exposure to stress.Psychoneuroendocrinology 15(5-6):317-328; Ulrich-Lai, Y. M, W. Xie, J.T. Meij, C. M. Dolgas, L. Yu, and J. P. Herman. 2006. Limbic and HPAaxis function in an animal model of chronic neuropathic pain. PhysiolBehav 88(1-2):67-76; the disclosures of which are herein incorporated byreference.

Evaluation may, in certain embodiments, include the identification orquantification of a stress phenotype biomarker or physiologicalparameter, e.g., to determine the presence of stress by identifying oneor more stress correlated biomarkers (e.g., cortisol) or physiologicalparameters (e.g., blood pressure), as described in Aardal, E. and A. C.Holm. 1995. Cortisol in saliva—reference ranges and relation to cortisolin serum. Eur J Clin Chem Clin 33(12):927-932; Abelson, K. S., B. Adem,F. Royo, H. E. Carlsson, and J. Hau. 2005. High plasma corticosteronelevels persist during frequent automatic blood sampling in rats. In Vivo19:815-819; Bayart, F., K. T. Hayashi, K. F. Faull, J. D. Barchas, andS. Levine. 1990. Influence of maternal proximity on behavioral andphysiological responses to separation in infant rhesus monkeys (Macacamulatta). Behav Neurosci 104(1):98-107; Blanc, J., M. L. Grichois, andJ. L. Elghozi. 1991. Effects of clonidine on blood pressure and heartrate responses to an emotional stress in the rat: A spectral study. ClinExp Pharmacol P 18(10):711-717; De Boer, S. F., S. J. Koopmans, J. L.Slangen, and G. J. van der Gugten. 1990. Plasma catecholamine,corticosterone and glucose responses to repeated stress in rats: Effectof interstressor interval length. Physiol Behav 47(6):1117-1124;Dhabhar, F. S., B. S. McEwen, and R. L. Spencer. 1993. Stress response,adrenal steroid receptor levels and corticosteroid-binding globulinlevels—a comparison between Sprague-Dawley, Fischer 344 and Lewis rats.Brain Res 616(1-2):89-98; Klosterman, L. L, J. T. Murai, and P. K.Siiteri. 1986. Cortisol levels, binding, and properties ofcorticosteroid-binding globulin in the serum of primates. Endocrinology118(1):424-434; McLaren, G. W., D. W. Macdonald, C. Georgiou, F.Mathews, C. Newman, and R. Mian. 2003. Leukocyte coping capacity: Anovel technique for measuring the stress response in vertebrates. ExpPhysiol 88(4):541-546; O'Connor, K. A., J. D. Johnson, M. K. Hansen, J.L. Wiesler-Frank, L. R. Watkins, and S. F. Maier. 2003. Peripheral andcentral proinflammatory cytokine response to a severe acute stressor.Brain Res 991:123-132; Randall, D. C., D. R. Brown, L. V. Brown, and J.M. Kilgore. 1994. Sympathetic nervous activity and arterial bloodpressure control in conscious rat during rest and behavioral stress. AmJ Physiol 267(5 Part 2):R1241-R1249; Windle, R. J., S. A. Wood, N.Shanks, S. L. Lightman, and C. D. Ingram. 1998. Ultradian rhythm ofbasal corticosterone release in the female rat: Dynamic interaction withthe response to acute stress. Endocrinology 139(2):443-450; thedisclosures of which are herein incorporated by reference.

In certain embodiments, the evaluation includes comparison to a controlor reference cell or organism of a like kind that is known not bestressed, such as is described in Faraday, M. M., K. H. Blakeman, and N.E. Grunberg. 2005. Strain and sex alter effects of stress and nicotineon feeding, body weight, and HPA axis hormones. Pharmacol Biochem Be80(4):577-589; van Bogaert, M. J., L. Groenink, R. S. Oosting, K. G.Westphal, J. van der Gugten, and B. Olivier. 2006. Mouse straindifferences in autonomic responses to stress. Genes Brain Behav5(2):139-149; the disclosures of which are herein incorporated byreference.

The stress condition of the cell or organism may be modulated prior toproduction of the biological material. For example, an unstressedphenotype may be produced in a cell or organism determined to exhibit astressed phenotype prior to production of the biological material. Anunstressed phenotype may be produced in the cell or organism using anyconvenient protocol, where the protocol employed may include modifyingthe environment of the cell or organism in some manner sufficient toproduce an unstressed phenotype. Protocols of interest include thosedescribed in the references incorporated herein, including NationalResearch Council. (2008) Recognition and Alleviation of Distress inLaboratory Animals. Washington, D.C.: The National Academies Press;Moberg G P, Mench J A (2000) The Biology of Animal Stress. BasicPrinciples and Implications for Animal Welfare. Wallingford: CABIPublishing; and Morton, D. B. and P. H. M. Griffiths. 1985. Guidelineson the recognition of pain, distress and discomfort in experimentalanimals and an hypothesis for assessment. Vet Record 116(16):431-436.The term “environment” refers broadly to the overall set of conditionsin which the cell or organism is present, including temperature, light,energy source availability, etc. As such, environmental modulation canbe accomplished in a variety of different ways, including but notlimited to: changing the temperature of the cell or organism, changingthe light experienced by the cell or organism, changing the food intakeof the cell or organism, administering a stress relieving agent to thecell or organism, etc. In certain embodiments, the unstressed cell ororganism is produced by changing the nutritional intake of the cell ororganism e.g., by modifying food intake of the cell or organism, such asby restricting or increasing the caloric intake of the cell or organism,by administering a certain kind of stress reducing food to the cell ororganism (e.g., an antixenohormetic agent or organism), etc. In certainembodiments, producing an unstressed phenotype in the cell or organismincludes confirming that the cell or organism has obtained theunstressed phenotype of interest, e.g., by comparing the cell ororganism to a control or reference. As reviewed above, the unstressedphenotype that is produced may vary greatly, e.g., from appearance tobehavior to certain unstressed genomic and/or proteomic profiles.

Because cells may be derived or harvested from an organism, in certainaspects the organism itself is first evaluated for the presence orabsence of a stressed phenotype. As described above, an organism may beacquired by any convenient means, including by purchasing from acommercial source that sells organisms. Prior to harvesting or derivingthe cells, the organism can be evaluated for the presence or absence ofa stressed phenotype, using the approaches as reviewed above. If astressed phenotype is identified, an unstressed phenotype may beproduced, e.g. using the approaches outlined above, prior to harvestingor deriving the cells from the organism. The cells may then be used toproduce a biological product. In certain aspects, the cells may also beevaluated to determine the presence or absence of a stressed phenotype,and an unstressed phenotype may be produced if a stressed phenotype isdetected, following the guidance and methods reviewed above.

The evaluation of the cells or organism to determine the presence orabsence of a stressed phenotype may occur prior to, during, and/orsubsequent to the production of the biological material. For example,the evaluation may occur immediately prior to production of the materialor at some time prior to production, e.g., about 1 month or less priorto production, 1 week or less prior to production, about 1 day or lessprior to production, about 12 hours or less prior to production, about 6hours or less prior to production, about 1 hour or less prior toproduction, about 30 minutes or less prior to production, about 10minutes or less prior to production, about 5 minutes or less prior toproduction, etc. The evaluation may occur subsequent to the start ofproduction, e.g. about 5 minutes or longer after the start ofproduction, about 10 minutes or longer after the start of production,about 30 minutes or longer after the start of production, about 1 houror longer after the start of production, about 6 hours or longer afterthe start of production, about 12 hours or longer after the start ofproduction, about 1 day or longer after the start of production, about 1week or longer after the start of production, etc. Evaluation mayinclude a single assessment or multiple assessments of the cell ororganism, specifically including assessments given before, during,and/or after production.

The methods of producing biological materials from the cells and/ororganisms may be made will vary based upon, for example, the nature ofthe cell or organism, the nature of the biological material, thepresence or absence of a stressed phenotype, and other factors readilyascertainable to those of skill in the art. In many aspects, methods ofproducing a biological material involve culturing a cell containing cellcontaining nucleic acid encoding at least a portion of the biologicalproduct, under conditions allowing for expression of the biologicalproduct; and recovering the biological product. The nucleic acid may beendogenous, or may be exogenous and be introduced into the cell ororganism using methods known to those of skill in the art.

For example, standard recombinant methods can often be used forproduction of a biological product, such as an antibody. For instance,nucleic acids encoding light and heavy chain variable regions of theantibody, optionally linked to constant regions, may be inserted intoexpression vectors. The light and heavy chains can be cloned in the sameor different expression vectors. The DNA segments encodingimmunoglobulin chains are operably linked to control sequences in theexpression vector(s) that ensure the expression of immunoglobulinpolypeptides. Expression control sequences include, but are not limitedto, promoters (e.g., naturally-associated or heterologous promoters),signal sequences, enhancer elements, and transcription terminationsequences. The expression control sequences can be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells (e.g., COS or CHO cells). Once the vector has beenincorporated into the appropriate host, the host is maintained underconditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the antibodies.

Moreover, suitable promoter and enhancer elements are known in the art.For expression in a bacterial cell, suitable promoters include, but arenot limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. Forexpression in a eukaryotic cell, suitable promoters include, but are notlimited to, light and/or heavy chain immunoglobulin gene promoter andenhancer elements; cytomegalovirus immediate early promoter; herpessimplex virus thymidine kinase promoter; early and late SV40 promoters;promoter present in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters.

In some embodiments, e.g., for expression in a yeast cell, a suitablepromoter is a constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1promoter, and AOX1 (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Suitable promoters for use in prokaryotic host cells include, but arenot limited to, a bacteriophage T7 RNA polymerase promoter; a trppromoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tachybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lacpromoter; a trc promoter; a tac promoter, and the like; an araBADpromoter; in vivo regulated promoters, such as an ssaG promoter or arelated promoter (see, e.g., U.S. Patent Publication No. 20040131637), apagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93;Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter(Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like (see,e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al.(2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter(see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); astationary phase promoter, e.g., a dps promoter, an spy promoter, andthe like; a promoter derived from the pathogenicity island SPI-2 (see,e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al.(2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g.,Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter(see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. andHeinemann, U. (eds), Topics in Molecular and Structural Biology,Protein—Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp.143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. AcidsRes. 12:7035); and the like. Suitable strong promoters for use inprokaryotes such as Escherichia coli include, but are not limited toTrc, Tac, T5, T7, and P_(Lambda.) Non-limiting examples of operators foruse in bacterial host cells include a lactose promoter operator (LacIrepressor protein changes conformation when contacted with lactose,thereby preventing the LacI repressor protein from binding to theoperator), a tryptophan promoter operator (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind to the operator), and a tac promoteroperator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci.U.S.A. 80:21-25).

Embodiments of the invention also include methods of screening acandidate compound for its ability to modulate the stress condition of acell or organism. By “screening” is meant assessing or evaluating anagent for its ability to modulate (i.e., change) a stress phenotype in acell or organism that may be used to produce a biological material. Assuch, methods include identifying whether an agent enhances or reduces,including inhibits, a stress phenotype in a cell or organism. Inpracticing these embodiments of the invention, the methods may includecontacting a candidate compound with an organism exhibiting a knownstress phenotype (such as an organism identified as described above);and evaluating the organism to determine any change in the stressphenotype to identify the compound for its ability to modulate a stressphenotype.

The candidate agent may be contacted with the cell or organism using anyconvenient protocol, e.g., by placing the agent in the nutrient mediumof the cell or organism, by administering the agent to the cell ororganism, etc. A large number of different types of compounds may beevaluated for their ability to modulate a stress phenotype. Compoundsthat may be evaluated encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Compounds may include functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andmay include at least an amine, carbonyl, hydroxyl or carboxyl group,such as at least two of the functional chemical groups. The compoundsmay include cyclical carbon or heterocyclic structures and/or aromaticor polyaromatic structures substituted with one or more of the abovefunctional groups. Compounds of interest are also found amongbiomolecules including, but not limited to: peptides, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof.

Compounds of interest may be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. New potential agents may also be created usingmethods such as rational drug design or computer modeling.

Following contact of the compound composition with the cell or organism,the effect of the compound on the cell or organism is determined. Theeffect of the compound on the organism may be determined by evaluatingone or more of a number of different phenotypic parameters. Phenotypicparameters that are evaluated in a given assay of the subject inventionmay vary widely depending, at least in part, on the nature of the cellor organism being evaluated. Phenotypic parameters that may be evaluatedin any given assay include one or more of the following: (1) viability;(2) morphological defects; and (3) fecundity. Specific parameters thatmay be evaluated include one or more of: (1) lethal dose, e.g. LD50,LD10 etc.); (2) growth defects; (3) sterility effect dose; (4)developmental defects; (5) neurologic impairment; (6) life-spanmodulation, e.g. life span enhancing or shortening; and the like. Ofparticular interest in certain embodiments is the assessment of whetherthe cell or organism has reverted to a non-stressed state.

In addition to the above parameters that can be evaluated in the subjectmethods, the gene expression levels of the test cell or organism can beassayed, e.g. gene expression levels in treated larva, pupa, and/orflies can be evaluated. The genes can be from “housekeeping” genes thatprovide basic metabolic information to developmental and tissue specificgenes to gauge which tissue or cell type is affected and when. A varietyof different gene expression protocols, including arrays basedprotocols, are known to those of skill in the art, including thosedescribed in: EP 0 328 829 B1 and U.S. Pat. Nos. 5,468,613; 5,580,726;5,599,672; 5,512,462; 5,162,209 and 5,162,209, the disclosures of whichare herein incorporated by reference. Methods of analyzing differentialgene expression are also described in Maniatis, et al., MolecularCloning, A Laboratory Manual, (Cold Spring Harbor Press, Cold SpringHarbor, NY) (1989); Nucleic Acid Hybridization, A Practical Approach(Hames, B. D., and Higgins, S. J. eds, IRL Press, Oxford) (1985); WO95/21944; Chalifour, et al., Anal. Biochem. (1994) 216: 299 304; Nguyenet al., Genomics (1995) 29: 207 216; Pietu et al., Genome Res. (1996) 6:492 503; and Zhao et al., Gene (1995) 166: 207 213.

Patents and patent applications describing methods of genomic expressionanalysis include, but are not limited to: U.S. Pat. Nos. 5,143,854;5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980;5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800,992. Alsoof interest are U.S. Pat. Nos. 6,656,740; 6,613,893; 6,599,693;6,589,739; 6,587,579; 6,420,180; 6,387,636; 6,309,875; 6,232,072;6,221,653; and 6,180,351.

Also of interest are proteomic analysis assays, e.g., where arrays ofpolypeptide binding agents are employed. Where the arrays are arrays ofpolypeptide binding agents, e.g., protein arrays, specific applicationsof interest include analyte detection/proteomics applications, includingthose described in U.S. Pat. Nos. 4,591,570; 5,171,695; 5,436,170;5,486,452; 5,532,128 and 6,197,599 as well as published PCT applicationNos. WO 99/39210; WO 00/04832; WO 00/04389; WO 00/04390; WO 00/54046; WO00/63701; WO 01/14425 and WO 01/40803—the disclosures of which areherein incorporated by reference.

The effect of the compound on the particular physical parameter orparameters being evaluated may be determined manually or robotically,such that in many embodiments determination of the effect of thecompound on the cell or organism is accomplished via an automatedprocedure.

The effect of the compound on the phenotypic parameter or parameters isthen related to the ability of the compound to modulate the stresscondition of the cell or organism.

Also provided are methods of using cells or organisms known to haveunstressed phenotypes to produce a product. By known unstressedphenotype is meant that the stress state of the cell or organism ispredetermined. As such, in some way the stress condition of the cell ororganism has been evaluated and is known to be unstressed.

EXPERIMENTAL

As can be appreciated from the disclosure provided above, the presentdisclosure has a wide variety of applications. Accordingly, thefollowing examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Those of skill in the art will readily recognizea variety of noncritical parameters that could be changed or modified toyield essentially similar results. Thus, the following examples are putforth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use the presentinvention, and are not intended to limit the scope of what the inventorsregard as their invention nor are they intended to represent that theexperiments below are all or the only experiments performed. Effortshave been made to ensure accuracy with respect to numbers used (e.g.amounts, temperature, etc.) but some experimental errors and deviationsshould be accounted for.

EXAMPLE 1 Production of a Biological Product from Cells

Vectors encoding a known antibody are incorporated into CHO host cells.At various timepoints, the CHO host cells are assayed to detect thepresence or absence of a stressed phenotype. The cells are then dividedinto two groups, a control group and an experimental group. Theenvironment of the cells of the experimental group is modulated byaltering the temperature and nutritional intake of the cells. Allcells—those of both the control group and the experimental group—aremaintained under conditions suitable for high level expression of thenucleotide sequences. Antibodies are subsequently collected andpurified. The yield and efficacy of the antibodies is assayed.

EXAMPLE 2 Production of a Biological Product from an Organism

Transgenic goats that produce an antibody are divided into multiplegroups. At various, the goats are assayed to detect the presence orabsence of a stressed phenotype. The goats are administered differentdiets for four weeks. Antibodies produced during this time are assayedfor efficacy, and yields are calculated. This enables identification ofspecific stress modulating conditions that produce antibodies that havethe desired safety, efficacy, and/or yield.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this disclosure that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A method for producing a biological product from a cell, the methodcomprising: culturing the cell containing nucleic acid encoding at leasta portion of the biological product, under conditions allowing forexpression of the biological product, and wherein the conditionsmodulate a stressed phenotype of the cell; and recovering the biologicalproduct.
 2. The method according claim 1, comprising evaluating the cellto identify the presence or absence of a stressed phenotype.
 3. Themethod according to claim 2, wherein evaluating the cell to identify thepresence or absence of a stressed phenotype is performed prior torecovering the biological product.
 4. The method according claim 1,comprising producing an unstressed phenotype in the cell.
 5. The methodaccording to claim 4, wherein producing an unstressed phenotypecomprises changing the cell's nutritional intake.
 6. The methodaccording to claim 4, wherein producing an unstressed phenotypecomprises changing the cell's environment.
 7. The method according toclaim 6, wherein the unstressed phenotype is produced by changing thecell's temperature or the cell's exposure to light.
 8. The methodaccording to claim 4, comprising administering to the cell an agent toreduce stress.
 9. (canceled)
 10. The method according to claim 4,wherein the evaluating comprises visually inspecting the cell.
 11. Themethod according to claim 4, wherein the evaluating comprises genomicassessment or proteomic assessment of the cell.
 12. The method accordingto claim 1, wherein the cell is mammalian.
 13. (canceled)
 14. The methodaccording to claim 1, wherein the cell is obtained from an organismhaving an unstressed phenotype.
 15. The method according to claim 14,wherein the unstressed phenotype is produced in the organism by changingthe organism's nutritional intake.
 16. The method according to claim 14,wherein the unstressed phenotype is produced in the organism by changingthe organism's environment.
 17. The method according to claim 16,wherein the unstressed phenotype is produced in the organism by changingthe organism's temperature or the organism's exposure to light. 18-19.(canceled)
 20. The method according to claim 14, comprising evaluatingthe organism to identify the unstressed phenotype. 21-24. (canceled) 25.The method according to claim 1, wherein the biological product is anantibody.
 26. (canceled)
 27. The method according to claim 1, whereinthe biological product is a peptide.
 28. A method for producing abiological product, the method comprising: evaluating an organism toidentify the presence or absence of an unstressed phenotype; extractinga plurality of cells from the organism; introducing nucleic acidencoding at least a portion of the biological product into the pluralityof cells; culturing the cells under conditions allowing for expressionof the biological product; and recovering the biological product. 29-42.(canceled)
 43. A method for producing a product from an organism havingan unstressed phenotype, the method comprising: evaluating an organismto identify the presence of a stressed phenotype; producing anunstressed phenotype in the organism; maintaining the organism underconditions allowing for production of the product; and recovering theproduct. 44-52. (canceled)